Role of the Efflux Pumps in

Antimicrobial Resistance in E. coli

PatrickPlsiat
BacteriologyDepartment
TeachingHospital
Besanon,France

ANTIBIOTIC

TARGET

Bacterial targets for antibiotics

Chromosome
Cell wall
Ribosomes

Cytoplasmic membrane

Main resistance mechanisms to drugs

Inactivation
Modification
ANTIBIOTIC
Efflux
Impermeability
Protection
TARGET
Substitution
Amplification

Reduced affinity
- mutations
- recombinaisons
- enzymatic modification

Gram-negative species with known efflux systems

Escherichia coli
Salmonella Typhimurium
Shigella dysenteriae
Klebsiella pneumoniae
Enterobacter aerogenes
Serratia marcescens
Proteus vulgaris
Citrobacter freundii...

Bacteroides fragilis...

Pseudomonas aeruginosa
Pseudomonas putida
Burkholderia cepacia
Burkholderia pseudomallei
Stenotrophomonas maltophilia
Alcaligenes eutrophus...

Haemophilus influenzae
Campylobacter jejuni
Helicobacter pylori
Vibrio parahaemolyticus
Vibrio cholerae
Neisseria gonorrhoeae...

Efflux mechanisms: practical implications

Do efflux systems produce clinically relevant levels of

resistance ?

Does the expression of drug transporters somewhat impair the

virulence of bacterial pathogens ?

What is the prevalence of efflux systems relative to other

resistance mechanisms among the clinical isolates ?

How to recognize efflux mutants in laboratory practice ?

What recommendations can be made to the physician for the

treatment of patients infected with mdr strains ?

Intracellular accumulation

Drug accumulation experiments

S
ATP
glucose

R
CCCP

Time

Structure of bacterial efflux systems

One component systems

Mostly in Gram positive species (except Tet...)
A single transporter protein in the cytoplasmic membrane
Determines the substrate specificity and resistance

Three component (tripartite) systems

Exclusively in Gram negative species (GNB)

A transporter protein

A periplasmic adaptor lipoprotein

A outer membrane channel protein

Energy sources

Antiporters
PMF transporters (proton motive force)
Na+-antibiotic antiporters

ABC transporters
ATP binding cassette pumps
Hydrolysis of ATP into ADP + Pi
Mostly in Gram positive species

PMF transporters

Major Facilitator Superfamily (MFS)

Drug efflux
12 TMS transporters
14 TMS transporters
Active uptake/export
sugars...
amino acids, secondary metabolites...

Small Multidrug Resistance Family (SMR)

Resistance/Nodulation Cell Division Family (RND)

4 TMS transporters
12 TMS transporters

Multi Antimicrobial Extrusion Family (MATE)

12 TMS transporters

Structure of drug efflux systems

antibiotic

antibiotic
H+

Na+
H+

ATP

MFS, SMR

MATE

ADP

ABC

RND, MFS, ABC

Fernandez-Recio J. et al. FEBS 2004, 578: 5-9

Murakami S. et al. Nature 2002, 419: 587

Murakami S. et al. Curr Opinion Struct. Biol. 2003, 13: 443

Murakami S. et al. Curr Opinion Struct. Biol. 2003, 13: 443

Efflux systems in E. coli

Chromosomally encoded pumps

37 putative drug transporters: 19 MFS, 3 SMR, 7 RND, 7 ABC,
1 MATE
20 pumps are able to transport toxic/antibiotic molecules
15-17 pumps may provide with some resistance to antibiotics when
overproduced from cloned genes (Nishino K et al. J. Bacteriol. 2001)
Upregulation of a single pump may result in increased drug efflux

Acquisition of exogenous pump encoding genes

Genes carried by mobile elements (plasmids, transposons,
integrons)

Efflux pumps coded by mobile genetic elements

Species

System

E. coli
E. coli
E. coli
E. coli

TetA/B/E
CmlA
Flo
OqxAB-TolC

Family
MFS
MFS
MFS
RND

Substrates
Tc, Min
Cmp
Cmp, Flo
Olaquindox, Cmp

Tc: tetracycline; Min: minocycline; Cmp: chloramphenicol; Flo: florfenicol

Efflux pumps of MFS, MATE, SMR, or ABC family

Species

System

E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli

EmrAB-TolC
Bcr
MdfA
MdtG
MdtH
MdtL
MdtM
NorE
EmrE
MdtJK
MacAB-TolC

Family
MFS
MFS
MFS
MFS
MFS
MFS
MFS
MATE
SMR
SMR
ABC

Substrates

Genes

Nal
Tc, Km, Fos
Tc, Rif, Cmp, Ery, Neo, Fq...
Fos
Fq
Cmp
Cmp, Fq
Cmp, Fq, Fos, Tmp
Tc
Nal, Fos
Ery

C
C
C
C
C
C
C
C
C
C
C

Nal: nalidixic acid; Tc: tetracycline + glycylcyclines; Km: kanamycin; Fos: fosfomycin; Rif: rifampicin; Cmp: chloramphenicol;
Ery: erythromycin; Neo: neomycin; Fq: fluoroquinolones; Tmp: trimethoprim

Efflux pumps of the RND family

Bacteria

System

Substrates

E. coli

AcrAB-TolC1

Fq, -lactams3, Tc, Cmp, Nov, Ery, Fus, Rif

E. coli

AcrEF-TolC2

Fq, -lactams3, Tc, Cmp, Nov, Ery, Fus, Rif

E. coli

AcrD2-AcrA-TolC

AGs, Ery, PolyB

E. coli

CusAB-?2

Fos

E. coli

MdtABC-TolC2

Fq

E. coli

MdtEF-TolC2

Ery

P. aeruginosa

MexAB-OprM1

Fq, -lactams1, Tc, Cmp, Nov, Ery, Fus, Tm...

P. aeruginosa

MexCD-OprJ2

Fq, 3rd GC, Tc, Cmp, Ery, Tmp

P. aeruginosa

MexEF-OprN2

Fq, Cmp, Tmp

P. aeruginosa

MexXY2-OprM

Fq, AGs, 3rdGC, Ery, Tc

N. gonorrhoeae

MtrCDE1

Tc, Cmp, -lactams1, Ery, Fus, Rif...

Fq: (fluoro)quinolones; Tc: tetracycline; Cmp: chloramphenicol; Nov: novobiocin; Ery: erythromycin; Fus: fusidic acid; Rif: rifampicin;
AGs: aminoglycosides; PolyB: polymyxin B; Tmp: trimethoprim; Sulf: sulfamethoxazole; 3 rdGC: cefepime, cefpirome. 1 expressed
constitutively in wild type cells, 2 inducible expression, 3 except imipenem.

Induction of acrAB-tolC expression

tetracycline
chloramphenicol
salicylate-acetylsalicylate
benzoate
stress...

MarROAB

SoxSR
Rob

Porin OmpF
TolC
AcrAB
EmrAB
Other proteins

Marregulon

oxidative stress
bile salts

tetracycliner
chloramphenicolr
quinolonesr
erythromycinr
solvants, pine oil...

Overexpression of acrAB and mtrCDE operons

_
MarA

acrA

MarR
_

+
-

(MppA)

SoxS

SoxR

acrB

acrR

MtrA
+
-

mtrC

mtrD

mtrE

mtrR
mutations mdr

*
*
*
*

*
*
*
*
*
*
*

WebberM.etal.Antimicrob.AgentsChemother.2001,45:1550

Systems MtrCDE and FarAB in N. gonorrhoeae

Antibiotics

wild type

CDE++

CDE-

FarAB-

Penicillin G

0.008

0.032

0.008

nd

Erythromycin

0.25

1-2

0.06

0.25

Tetracycline

0.25

0.5

nd

nd

Rifampicin

0.06

0.25

0.015

nd

Linoleic acid

1600

nd

25 - 50

50

Palmitic acid

100

nd

12.5

12.5

System AcrAB-TolC in E. coli

Antibiotics

wild type

AcrAB++

AcrAB-

Nalidixic acid

4-6

8.5 - 32

0.6

Norfloxacin

0.025 - 0.1

0.3 - 1.25

nd

Ofloxacin

0.06 - 0.07

0.25 - 0.3

nd

Ciprofloxacin

0.02

0.15

nd

Ampicillin

2-4

5-6

0.6 - 2

128 - 256

> 512

<2-8

Tetracycline

1.25 - 3

5 - 16

0.25 - 0.3

Chloramphenicol

4 - 7.5

10 - 28

0.6

Erythromycin

System MexAB-OprM in P. aeruginosa

Antibiotics

wild type

MexAB++

MexAB-

Norfloxacin

0.25 - 1

2-4

0.05 - 0.25

Ofloxacin

0.4 - 1

1.6 - 8

0.025 - 0.05

Ciprofloxacin

0.03 - 0.25

0.4 - 1.6

0.012 - 0.03

Carbenicillin

12.5 - 64

50 - 256

0.4 - 1

Aztreonam

1.6 - 4

12.5 - 32

0.1 - 0.2

Ceftazidime

0.4 - 2

1.6 - 8

0.2 - 0.4

Cefepime

0.8 - 2

3-4

0.1 - 0.5

Meropenem

0.2 - 0.5

0.8 - 2

0.1 - 0.2

Tetracycline

6.25 - 16

25 - 64

0.2 - 1.2

Chloramphenicol

12.5 - 32

100 - 512

0.8 - 2

Interplays between resistance mechanisms in GNB

Outer membrane
permeability

Other mechanisms

Active efflux

Efflux/target double mutants of E. coli

Genotype/Phenotype

Oflo

Cipro

wild type AG100

0.03

0.015

AcrAB++

0.125

0.06

gyrA (Asp87->Gly)

0.25

0.25

gyrA (Asp87->Gly; Ser83->Leu)

gyrA (Asp87->Gly), AcrAB++

gyrA (Asp87->Gly), AcrAB-

0.06

0.03

Oethingeretal.Antimicrob.AgentsChemother.2000,44:1013

Therapeutic implications of efflux systems

Resistance levels conferred by intrinsic pumps

Low to moderate drug resistance (MIC x 2 - 16)
Clinical significance
Lack of clinical data !
Poor response to treatment when the concentrations of
antibiotics are low at the infection site (insufficient dosage,
inappropriate drug, abcess...)
Increased emergence of target mutants ?

Emergence of efflux mutants under treatment

Cross resistance to structurally unrelated molecules
Role of fluoroquinolones

PK/PD Monte Carlo

MIC (mg/L)

Treatment
Drug

Ciproflox.

total daily dosage

(mg)

unitary dose interval

(hours)

1200

1600

2400

Levoflox.

500

1000

24

12

Target Attainment Rate (%)

Cmax/MIC > 10

AUC/MIC > 125

0.12

66

87

0.25

0.12

66

90

0.25

12

0.12

98

100

0.25

60

85

0.5

4.2

3.7

0.5

70

40

0.5

72

72

DupontP.etal.J.Antimicrob.Chemother.2005

Efflux mutants, are they virulent ?

Clinical experience
Many examples of mdr isolates recovered from clinical specimens
(blood, urine, sputums)

Other considerations
marA disruption mutants of S. Typhimurium remain fully virulent in a
murine BALB/c infection model (Sulavik, J. Bacteriol. 1997, 179: 1857)
First step fluoroquinolone resistant mutants with mutations in gyrA,
gyrB or marOR do not display significant loss of fitness (in vitro
competition experiments, experimental urinary tract infection in
mouse) (Komp Lindgren P., AAC 2005, 49: 2343)
Role of secondary mutations ?

How to characterize efflux mechanisms

Plasmid or transposon encoded efflux systems

Multiresistance phenotype
Detection of efflux gene(s): PCR, nucleic probes

Upregulation of intrinsic efflux systems

Protein levels

Western blotting of membrane extracts with specific antibodies

mRNA levels

Northern blot, MacroArray, MicroArray

Real Time RT-PCR (Light Cycler, Taq Man, I Cycler)

Intracellular accumulation of antibiotics

[3H] ou [14C] radiolabeled or fluorescent compounds (BET,

acriflavine)

Sequencing of regulatory genes

Efflux inhibitors

PhenylArginylNnaphtylamide